Chitinases are enzymes that hydrolyze chitin which is a constituent of exoskeleton of insects and crustaceans as well as cell walls of moulds, and widely distributed in microorganisms and plants.
Plants have 5 kinds of chitinases classified as classes I-V, amongst those classes I, II, IV and V chitinases have an ability of decomposing cell walls of moulds but lack an ability of decomposing cell walls of bacteria. On the other hand, the class III chitinase has an ability of decomposing cell walls of bacteria but lacks an ability of decomposing cell walls of moulds.
The present invention relates to a complementary DNA for a novel rice chitinase having a lytic activity against both of moulds and of bacteria as well as to utilization thereof.
Agriculture sustains a great loss by a decrease in yield of harvest because of an infection by plant pathogenic microorganisms. As a means for prevention of the infection and inhibition of enlargement of the infection by these microorganisms, spraying of germicides has been carried out.
In recent years, however, there is a demand for the development of techniques for preventing and exterminating against the infection of pathogens without relying on chemical substances, because of anxiety for influences to the environment and to the human body by these germicides.
Plants have their unique preventing and exterminating mechanisms against invading pathogenic microorganisms. Specifically, when plants become aware of infection by a pathogenic microorganism, they protect themself or their plant bodies from the infection by producing antimicrobial agents such as a compound called phytoalexin, pathogenicity related protein (PR protein) and so on.
The PR protein includes, as enzymes having the clearest action mechanism against microorganisms, chitinases and .beta.-1, 3-glucanases that hydrolyze chitin and .beta.-1, 3-glucan that constitute cell walls of moulds.
As described above, plants have 5 kinds of chitinases classified as classes I-V, amongst those classes I, II, IV and V chitinases have an antifungal activity inhibiting growth of moulds. These chitinases act on the growing of hypha at which synthesis of cell wall is particularly active. As the result, the growing cells are destructed and extension of hypha is inhibited.
Amongst them, the classes I and IV enzymes having a chitin binding region have particularly high antifungal activity, and are attracting attention as a gene to be introduced for development of crop plants being resistant against the pathogenic moulds.
Under such circumstances, researches have been conducted to develop crop plants being resistant against the pathogenic moulds by introducing a complementary DNA for a chitinase into the plants.
While, however, the chitinase encoded by said introduced gene can certainly decompose the cell walls of the pathogenic moulds, they do not have a lysozyme activity decomposing the cell walls of bacteria. Therefore, even if a chitinase encoded by said gene is introduced into the crop plants, the plants are not provided with the resistance against the pathogenic bacteria. Accordingly, such a character is insufficient for creating resistant crop plants.
On the other hand, it has long been known that plants have an enzyme having a lysozyme activity. In addition, it has also been known that hevamine existing in latex of rubber tree and some class III chitinase have a lysozyme activity lysing the cell walls of bacteria. No report, however, has been presented for a complementary DNA and a gene relating to hevamine. In addition, no complementary DNA for a chitinase having a lysozyme activity has been known.
Thus, until now, no complementary DNA for a chitinase having both of the antifungal activity and the lysozyme activity has been known; i.e. it has not been demonstrated by expressing a protein that a complementary DNA encodes a chitinase having both of the antifungal activity and the lysozyme activity.
The purpose of the present invention is to demonstrate a possibility of developing a recombinant crop plant having an acquired resistance against a wide range of pathogenic microorganisms by identifying a complementary DNA encoding a novel chitinase capable of lysing cell walls of both moulds and bacteria and introducing the sequence thereof into a plant.
After conducting extensive studies in order to attain the above purpose, the present inventors have successfully isolated from rice plant a complementary DNA encoding a chifinase having a lysing activity to both of the moulds and bacteria by a method described below.
Thus, the present inventors have determined the complete nucleotide sequence of a complementary DNA contained in S4960 clone isolated by the Rice Genome Project, and have revealed that said clone contains a complementary DNA having 1,109 bp encoding a protein having a molecular weight of 32,260 Dalton and comprising 305 amino acid residues. They have considered that said protein is probably a chitinase because the amino acid sequence thereof have 68% homology with the sequence of hevamine which is the class III chitinase contained in the latex of rubber tree.
Subsequently, in order to construct an expression system for said protein, they have conducted researches for constructing the expression system using several kinds of microorganisms as hosts. As the result, they discovered that said protein could be efficiently expressed with secretion when Pichia pastoris, a kind of yeast, is used as the host.
In addition, they have purified said protein produced in Pichia pastoris and demonstrated that the purified protein is a chitinase capable of decomposing glycol chitins and chito-oligosuccharides. Furthermore, they have demonstrated that the purified protein exhibit an antifungal activity against Trichoderma reesei, a mould, and simultaneously, a lysozyme activity lysing cells of Micrococcus lysodeikticus, a bacterium. The present invention has been completed based on these findings.